(a) Field of the Invention
The invention relates to peptide inhibitors of thrombin that can be used as potent anticoagulants, and that are composed of natural amino acids or that can be made by recombinant techniques.
(b) Description of Prior Art
A wide range of medical conditions including atherosclerosis, infections and cancer can trigger thrombotic complications, leading to heart attack, stroke, deep-vein thrombosis, or pulmonary embolism (Libby, P. (2002) Nature 420, 868-874; Levi, M., Keller, T. T., van Gorp, E., and ten Cate, H. (2003) Cardiovasc. Res. 60, 26-39; Opal, S. M. and Esmon, C. T. (2003) Crit Care 7, 23-38; Loynes, J. and Zacharski, L. (2003) Expert. Opin. Ther. Targets. 7, 399-404; and Schultz, M. J., Levi, M., and van der, P. T. (2003) Curr. Drug Targets. 4, 315-321). As such, thrombosis, or the aberrant formation of a blood clot, has been the single largest cause of human disability and death in the world. In many situations, it is the occlusive blood clot that is life-threatening for patients with atherosclerosis and related cardiovascular diseases (Libby, P. (2002) Sci. Am. 286, 46-55; Libby, P. (2002) Nature 420, 868-874; and Virmani, R., Burke, A. P., and Farb, A. (2001) Cardiovasc. Pathol. 10, 211-218) or on long-term anti-HIV treatments (Madamanchi, N. R., Patterson, C., and Runge, M. S. (2002) Arterioscler. Thromb. Vasc. Biol. 22, 1758-1760; and Zhong, D. S., Lu, X. H., Conklin, B. S., Lin, P. H., Lumsden, A. B., Yao, Q., and Chen, C. (2002) Arterioscler. Thromb. Vasc. Biol. 22, 1560-1566). Pathogenic blood coagulation or thrombosis aggravates the symptoms of chromic liver infections and underlines the lethality of many infectious diseases (Levi, M., Keller, T. T., van Gorp, E., and ten Cate, H. (2003) Cardiovasc. Res. 60, 26-39; Marsden, P. A., Ning, Q., Fung, L. S., Luo, X., Chen, Y., Mendicino, M., Ghanekar, A., Scott, J. A., Miller, T., Chan, C. W., Chan, M. W., He, W., Gorczynski, R. M., Grant, D. R., Clark, D. A., Phillips, M. J., and Levy, G. A. (2003) J. Clin. Invest 112, 58-66; and Opal, S. M. and Esmon, C. T. (2003) Crit Care 7, 23-38). Malignant cells have been found to constitutively express the procoagulant tissue factor, generating hypercoagulability in cancer patients (Agorogiannis, E. I. and Agorogiannis, G. I. (2002) Lancet 359, 1440; Lorenzet, R. and Donati, M. B. (2002) Thromb. Haemost. 87, 928-929; and Ornstein, D. L., Meehan, K. R., and Zacharski, L. R. (2002) Semin. Thromb. Hemost. 28, 19-28). These recent observations have attracted significant attention to the potential use of anticoagulants or antithrombotic agents as part of new treatment strategies for devastating human cancers (Loynes, J. and Zacharski, L. (2003) Expert. Opin. Ther. Targets. 7, 399-404; Kakkar, A. K. (2003) Cancer Treat. Rev. 29 Suppl 2, 23-26; Levine, M. N. (2003) Cancer Treat. Rev. 29 Suppl 2, 19-22; Lee, A. Y. (2003) Expert. Opin. Pharmacother. 4, 2213-2220; and Deitcher, S. R. (2003) J. Thromb. Thrombolysis. 16, 21-31) and infectious diseases (Marsden, P. A., Ning, Q., Fung, L. S., Luo, X., Chen, Y., Mendicino, M., Ghanekar, A., Scott, J. A., Miller, T., Chan, C. W., Chan, M. W., He, W., Gorczynski, R. M., Grant, D. R., Clark, D. A., Phillips, M. J., and Levy, G. A. (2003) J. Clin. Invest 112, 58-66; Opal, S. M. and Esmon, C. T. (2003) Crit Care 7, 23-38; Geisbert, T. W., Hensley, L. E., Jahrling, P. B., Larsen, T., Geisbert, J. B., Paragas, J., Young, H. A., Fredeking, T. M., Rote, W. E., and Vlasuk, G. P. (2003) Lancet 362, 1953-1958; Robertson, M. (2003) Drug Discov. Today 8, 768-770; and Schultz, M. J., Levi, M., and van der, P. T. (2003) Curr. Drug Targets. 4, 315-321). However, the current generation of antithrombotic agents, among which many are thrombin inhibitors, lacks the required efficacy/safety. and cost-effectiveness (Gresele, P. and Agnelli, G. (2002) Trends Pharmacol. Sci. 23, 25-32; Vorchheimer, D. A. and Fuster, V. (2002) Eur. Heart J. 23, 1142-1144; and Weitz, J. I. and Buller, H. R. (2002) Circulation 105, 1004-1011) for realizing the tremendous potential of anticoagulant therapy in many disease indications.
Blood coagulation is one of the best-characterized physiological responses that involve tightly-regulated cascades of protein-protein interaction and enzyme activation reactions (Mann, K. G. (1999) Thromb. Haemost. 82, 165-174; and Furie, B. and Furie, B. C. (1988) Cell 53, 505-518). The coagulation processes can be triggered by the exposure of blood to open air and/or upon injury of the vascular wall (e.g. at the sites of atherosclerotic lesions). The clotting of the free blood is the result of the so-called “intrinsic” coagulation pathway started by the activation of factors XII and XI. Blood clots formed in closed circulation are initiated by the “extrinsic” coagulation pathway through contact of blood with exposed tissue factors (TF) on injured blood vessels. The two pathways converge on the activation of the circulating coagulation factor X into the factor Xa enzyme, which in turn is assembled into a macromolecular enzyme-cofactor complex, called the prothrombinase, containing factor Xa, factor Va, calcium ions and a phospholipid surface (Mann, K. G. (1999) Thromb. Haemost. 82, 165-174). There also appears to be a third pathway of blood coagulation, in which factor Xa of the prothrombinase is replaced by a tissue-specific Xa-like protein, the fgl2/fibroleukin, induced by the invasion of pathogenic viruses (Marsden, P. A., Ning, Q., Fung, L. S., Luo, X., Chen, Y., Mendicino, M., Ghanekar, A., Scott, J. A., Miller, T., Chan, C. W., Chan, M. W., He, W., Gorczynski, R. M., Grant, D. R., Clark, D. A., Phillips, M. J., and Levy, G. A. (2003) J. Clin. Invest 112, 58-66; and Chan, C. W., Chan,. M. W., Liu, M., Fung, L., Cole, E. H., Leibowitz, J. L., Marsden, P. A., Clark, D. A., and Levy, G. A. (2002) J. Immunol. 168, 5170-5177). As well, factor Xa may be generated from the inactive precursor factor X by endogenous proteases secreted by invading microbes (Ntefidou, M., Elsner, C., Spreer, A., Weinstock, N., Kratzin,. H. D., and Ruchel, R. (2002) Mycoses 45 Suppl 1, 53-56; and Schoen, C., Reichard, U., Monod, M., Kratzin, H. D., and Ruchel, R. (2002) Med. Mycol. 40, 61-71). In all the coagulation pathways, the prothrombinase assembly rapidly converts prothrombin into active thrombin, the ultimate protease resulting from the coagulation cascades. Upon generation, thrombin induces formation of the fibrin clot from the soluble fibrinogen, activates the fibrin cross-linking factor XIII, stimulates the aggregation of platelets and catalyzes the conversion of factors V, VIII and XI into Va, VIIIa and XIa to amplify its own production. Thrombin also binds to the cell-anchored thrombomodulin to form the thrombin-thrombomodulin complex, which in turn activates protein C and the thrombin-activatable fibrinolysis inhibitor (TAFI), initiating the natural anticoagulation and anti-fibrinolysis pathways (Nesheim, M., Wang, W., Boffa, M., Nagashima, M., Morser, J., and Bajzar, L. (1997) Thromb. Haemost. 78, 386-391). The critical role of thrombin in making blood clots and in thrombotic diseases has stimulated in-depth studies on the structure and function of thrombin (Berliner, J. L. (1992) Thrombin: structure and function Plenum Press, New York) and the design of thrombin inhibitors as novel anticoagulants (Weitz, J. I. and Buller, H. R. (2002) Circulation 105, 1004-1011; Fenton, J. W., Ni, F., Witting, J. I., Brezniak, D. V., Andersen, T. T., and Malik, A. B. (1993) Adv. Exp. Med. Biol. 340, 1-13; and Song, J. and Ni, F. (1998) Biochem. Cell Biol. 76, 177-188).
The mainstays of clinical anticoagulant treatments are heparin, which is a cofactor of plasma-derived and naturally-occurring inhibitors of thrombin, and coumarins, such as arfarin, which antagonize the biosynthesis of vitamin K-dependent coagulation factors. Although effective and widely used, heparins and coumarins have practical limitations because their pharmacokinetics and anticoagulation effects are unpredictable, with the risk of many undesirable side effects, such as hemorraghing and thrombocytopenia resulting in the need for close monitoring of their use. Low-molecular-weight heparins (LMWHs) provide a more predictable anticoagulant response; however, discontinuation of heparin treatment can result in a thrombotic rebound due to the inability of these compounds to inhibit clot-bound thrombin. More seriously, heparins are involved in many aspects of cellular physiology (Kakkar, A. K. (2003) Cancer Treat. Rev. 29 Suppl 2, 23-26), making their long-term uses as anticoagulants plagued with potential side effects.
There is a need for direct thrombin inhibitors (DTI) that are able to target: (1) free and (2) clot-bound thrombin. Hirudin is a member of only the first class and is a naturally occurring polypeptide produced by the blood sucking leech hirudo medicinalis. Hirudin and its recombinant forms bind irreversibly to both the catalytic and substrate-recognition sites of thrombin. Other DTIs with lower molecular weights, such as DuP714, PPACK, and efegatran, have subsequently been developed, and these agents are better inhibitors of clot-bound thrombin and the thrombotic processes at sites of arterial damage. Such compounds inhibit thrombin by covalent attachment and can result in toxicity and nonspecific inhibition of other proteins. Eventually, further development of these small molecules was mostly abandoned. In recent years, the development of low molecular weight, active site-directed, and reversible DTIs has resulted in a number of highly potent and selective compounds, such as inogatran and melagatran. Ongoing clinical trails suggest that the binding characteristics of these low molecular weight DTIs may also result in bleeding complications.
The main problem of small molecular weight compounds is their limited specificity since thrombin belongs to the family of serine proteases and these compounds do not have strong specificity toward thrombin alone. Also available data so far indicate that the development of the small-molecule DTIs of thrombin is more time-consuming than other inhibitory molecules. For example, it has proven difficult to disrupt high-affinity and highly-specific protein-protein interactions by use of small-molecule inhibitors (Benard, V., Bokoch, G. M., and Diebold, B. A. (1999) Trends Pharmacol. Sci. 20, 365-370; Cochran, A. G. (2001) Curr. Opin. Chem. Biol. 5, 654-659; and Veselovsky, A. V., lvanov, Y. D., Ivanov, A. S., Archakov, A. I., Lewi, P., and Janssen, P. (2002) J. Mol. Recognit. 15, 405-422). On the other hand, in addition to natural proteins (e.g. antibodies), novel polypeptide ligands have been discovered and shown to possess the ability to interfere selectively with the targeted protein-protein interactions (Cochran, A. G. (2001) Curr. Opin. Chem. Biol. 5, 654-659; Veselovsky, A. V., Ivanov, Y. D., Ivanov, A. S., Archakov, A. I., Lewi, P., and Janssen, P. (2002) J. Mol. Recognit. 15, 405-422; Juliano, R. L., Astriab-Fisher, A., and Falke, D. (2001) Mol. Interv. 1, 40-53; and Sidhu, S. S., Fairbrother, W. J., and Deshayes, K. (2003) Chembiochem. 4, 14-25). Linking of weak-binding molecules to create bivalent or multivalent molecules (DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J., Ni, F., and Konishi, Y. (1990) J. Biol. Chem. 265, 21698-21703; and Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandran, K. L., and Fenton, J. W. (1990) Biochemistry 29, 7095-7101) has also emerged as a general strategy for the design of potent inhibitors of enzymes (Jahnke, W., Florsheimer, A., Blommers, M. J., Paris, C. G., Heim, J., Nalin, C. M., and Perez, L. B. (2003) Curr. Top. Med. Chem. 3, 69-80; and Shuker, S. B., Hajduk, P. J., Meadows, R. P., and Fesik, S. W. (1996) Science 274, 1531-1534), receptors (Jahnke, W., Florsheimer, A., Blommers, M. J., Paris, C. G., Heim, J., Nalin, C. M., and Perez, L. B. (2003) Curr. Top. Med. Chem. 3, 69-80; and Kramer, R. H. and Karpen, J. W. (1998) Nature 395, 710-713) and protein-protein interactions (Song, J. and Ni, F. (1998) Biochem. Cell Biol. 76, 177-188; Mammen, M., Choi, S. K., and Whitesides, G. M. (1998) Angew. Chem. Int. Ed. 37, 2754-2794; and Mourez, M., Kane, R. S., Mogridge, J., Metallo, S., Deschatelets, P., Sellman, B. R., Whitesides, G. M., and Collier, R. J. (2001) Nat. Biotechnol. 19, 958-961). Intervention of cellular and physiological processes with multivalent polypeptides in particular allows access to the built-in evolutionary specificity of naturally-occurring protein-protein interactions, potentially avoiding the non-specific binding or side effects often seen with small molecules. The synthetic compound bivalirudin is one example of the better DTIs with two covalently linked groups that bind to both the catalytic and substrate-recognition sites of thrombin.
“In late 1988, a research program was initiated at the Biotechnology Research Institute (BRI) on the design of tbrombiu inhibitors as antithrombotic agents. In this research program, a series of novel compounds (the Canadian version of Angiomax® (bivalirudin)) (Fenton, J. W., Ni, F., Witting, J. I., Breznialc, D. V., Andersen, T. T., and Malik, A. B. (1993) Adv. Exp. Med. Biol. 340, 1-13; Song, J. and Ni, F. (1998) Biochem. Cell Riot 76, 177-188; DiMaio, J., Konislik Y., U.S. Pat. No. 6,060,451; and CA 2,085,465) that mimic the multivalent action of biradin, a natural antithrombin from medicinal leeches were discovered. These early research efforts have started to pay off as the related bivalent peptide, bivalirudin or hiralog, mimicking the action of hirudin has recently proved its clinical efficacy (Weitz, J. I. and Buller, H. R. (2002) Circulation 105, 1004-1011; Hirsh, J. (2003) Thromnb. Res. 109Suppl 1, S1-S8; Salarn, A. M. (2003) Expert. Opin. Investig. Drugs 12, 1027-1033; and Wykrzykowska, J. J., Kathiresan, S., and Jang, I. K. (2003) J. Thromb. Thrombolysis 15, 47-57).”
“Thrombin inhibitors issued from the research program were also disclosed in WO99/19356. These peptide inhibitors contain two covalently-linked motifs that bind to a large surface area encompassing the catalytic active site and a protein recognition exosite of thrombin. One of these peptide molecules, P53, has an amino acid sequence of (d)F-P-R-P-Q-S-H-N-D-G-D-F-B-E-I-P-E-E-Y-L-Q (DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J, Ni, F., and Konishi, Y. (1990) J. Biol. Chem. 265, 21698-21703; U.S. Pat. No. 6,060,451; and CA 2,085,465), which inhibits human α-thrombin with a Ki of ˜2.8 nM. The clinically-tested peptide known as hirulog or bivalirudin has a sequence of (d)F-P-R-P-G-G-G-G-N-G-D-F-E-E-I-P-E-E-Y-L, which is also a strong inhibitor of human α-thrombin (Ki˜2.3 nM) (Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandran, K. L., and Fenton, J. W. (1990) Biochemistry 29, 7095-7101). Hirulog-8 was approved and adopted for clinical uses since January 2001 under the trademark, Angiomax® (bivalirudin). However, the cost of Angiomax® (bivalirudin) is prohibitive since it contains an amino acid residue in the (d)-configuration, i.e. (d)F or (d)Phe, requiring chemical synthesis, limiting broader clinical applications.”
The latest clinical experiences showed that uses of the current generation of antithrombotic agents, among which many are direct thrombin inhibitors, can cause prolonged systemic bleeding during anticoagulation and may be associated with rebound activation of coagulation and the re-occlusion of opened blood vessels after anticoagulant therapy (Gresele, P. and Agnelli, G. (2002) Trends Pharmacol. Sci. 23, 25-32; Vorchheimer, D. A. and Fuster, V. (2002) Eur. Heart J. 23, 1142-1144; and Weitz, J. I. and Buller, H. R. (2002) Circulation 105, 1004-1011). In Canadian patent application CA 2,340,461, Shen et al. replaced the d-Phe moiety at the N-terminus of Bivalirudin with a 12 natural amino acids sequence derived from the thrombin receptor. This peptide, containing the LDPR (SEQ ID NO:1) sequence, exhibited an improved safety/efficacy profile with reduced bleeding complications as compared to Hirulog, despite having a significantly decreased binding affinity to thrombin (Xue, M., Ren, S., Welch, S., and Shen, G. X. (2001) J. Vasc. Res. 38, 144-152; and Chen, X., Ren, S., Ma, M. G., Dharmalingam, S., Lu, L., Xue, M., Ducas, J., and Shen, G. X. (2003) Atherosclerosis 169, 31-40). The (d)Phe-Pro-Arg sequence of P53 or Hirulog can also be replaced by the natural sequence of human FpA, i.e. acetyl-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg (SEQ IS NO:2) as proposed by Fenton et al (Fenton, J. W., Ni, F., Witting, J. I., Brezniak, D. V., Andersen, T. T., and Malik, A. B. (1993) Adv. Exp. Med. Biol. 340, 1-13) and synthesized previously (U.S. Pat. No. 5,433,940). The bivalent conjugate of FpA has been used along with an N-terminal extension to include a binding moiety for integrins on platelets (Mu, R., Qin, Y., Cha, Y., and Jing, Q. (2002) Zhonghua Yi. Xue. Za Zhi. 82, 593-596).
It would thus be highly desirable to be provided with peptide inhibitors of thrombin with good binding affinity and composed of genetically-encodable natural amino acids, as these peptides can be expressed through recombinant DNA or used in gene therapy.